P6A (H-Ala-Arg-Pro-Ala-Lys-OH) (SEQ ID NO: 1), one of the products obtained from the degradation of fibrinogen, was used as the lead compound in our previous studies on thrombolysis agents (Zhao, M.; Peng, S. Q. J Prakt. Chem. 1999, 341, 668; Zhao, M.; Lin, N.; Wang, C.; Peng, S. Q. Bioorg Med Chem Lett. 2003, 13, 961). As essential part of the researches the metabolism of P6A arouses our interest. In the identification of the in vivo metabolites the solution of P6A in normal saline was injected into the tail vein of the mice. After 20 min the plasma was isolated from the whole blood of the mice. To the plasma perchloric acid was added in order to free all of the metabolites of P6A from the plasma protein, centrifuged to separate the upper layer as a clear solution, and injected into the HPLC column of the HPLC/ESI/MS. In the HPLC/ESI/MS analysis all the possible ions of the metabolites from P6A in vivo were monitored by ESI/MS. According to the trapped ions the corresponding sequences were H-Ala-Arg-Pro-Ala-OH (SEQ ID NO: 2) (FAB/MS, 414), H-Ala-Arg-Pro-OH (FAB/MS, 343), H-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 3) (FAB/MS, 471), and H-Pro-Ala-Lys-OH (FAB/MS, 315). In the isolation of the in vitro metabolite P6A was added to the mice blood directly, incubated at 37° C. for 20 min, and centrifuged at 0° C. to isolate the plasma. To the plasma perchloric acid was added to free all of the metabolites of P6A from the plasma protein, and centrifuged to separate the upper layer which was frozen and lyophilized. The resulted crystals were dissolved in water and injected into the column. The fractions corresponding to the main peaks were collected, frozen and lyophilized. The FAB/MS tests of the resulted 4 crystals gave the same sequences as that as mentioned above.
The euglobulin clot lysis activity of the isolated metabolites resulted from the in vitro assay indicated that for development of thrombolytic peptides the corresponding modifications may be interesting. In the previous modification of oligopeptides we demonstrated that the introduction of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid may result in enhancement of bioactivity (Lin, N.; Zhao, M.; Wang, C.; Peng, S. Q. Bioorg Med Chem Lett. 2002, 12, 585; WU, Y. F.; Zhao, M.; Wang, C.; Peng, S. Q. Bioorg Med Chem Lett. 2002, 12, 2331).
In the present invention the pseudopeptides consisted of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which was prepared according to Scheme 1, H-Ala-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 1), H-Gln-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 4), H-Lys-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 5), H-Asn-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 6), H-Ala-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 1), H-Ala-Arg-Pro-Ala-OH (SEQ ID NO: 2), H-Ala-Arg-Pro-OH, H-Arg-Pro-Ala-Lys-OH (SEQ ID NO: 3) and H-Pro-Ala-Lys-OH, which were prepared according to Scheme 2, were prepared. The thrombolitic activities of the pseudopeptides were observed in vitro and in vivo.

Scheme 1. Synthesis, esterification and acylation of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 3S-(2-Boc)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid benzyl ester. (a) sulfuric acid; (b) (BOC)2O; (c) Cs2CO3, C6H5CH2Br.

Scheme 3. Synthetic route for Boc-Ala-Arg(Tos)-Pro-Ala-OBzl (SEQ ID NO: 7), Boc-Ala-Arg(Tos)-Pro-OBzl, H-Ala-Arg-Pro-Ala-OH (SEQ ID NO: 2) and H-Ala-Arc,-Pro-OH. (Sequences shown above are SEQ ID NOS 7, 2, 7 and 7, respectively in order of appearance).